The present invention relates to a novel process for preparing 2-hydroxymethyl penems which are useful in the synthesis of penems having antibacterial activity. More particularly the invention relates to a process for the preparation of compounds of formula I: ##STR3## wherein R.sub.1 represents a hydroxy protecting-group and R.sub.2 represents a carboxy protecting group, which process comprises hydrolyzing a compound of formula II: ##STR4## wherein R.sub.1 and R.sub.2 are as defined above and R represents an alkyl, alkenyl or phenylalkyl group having from 1 to 18 carbon atoms, by means of an enzyme capable of selectively hydrolysing the ester group of the 2-substituent thereof.
Suitable hydroxy protecting groups which R.sub.1 may represent include p-nitrobenzyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl, trimethylsilyl, benzyl, p-bromophenacyl, triphenylmethyl and pyranyl groups. Preferred protecting groups are p-nitrobenzyloxycarbonyl, trimethylsilyl and pyranyl.
Suitable carboxy protecting groups which R.sub.2 may represents include (a) alkyl groups having from 1 to 6 carbon atoms, (b) haloalkyl groups having from 1 to 6 carbon atoms, (c) alkenyl groups having from 2 to 4 carbon atoms, (d) optionally substituted aryl groups, (e) optionally substituted aralkyl groups, the alkyl part thereof has from 1 to 6 carbon atoms, and (f) aryloxyalkyl groups. Examples of these groups are: (a) methyl, ethyl and t-butyl, (b) 2,2,2-trichloroethyl, (c) allyl, (d) phenyl and p-nitrophenyl, (e) benzyl, p-nitrobenzyl, p-methoxybenzyl, diphenylmethyl and di-(o-nitrophenyl)-methyl and (f) phenoxymethyl. Other carboxy protecting groups include acetonyl and trimethylsilyl groups. Still other protecting groups are residues which are known to be hydrolyzed in vivo and which have favorable pharmacokinetic properties such as acetoxymethyl, pivaloyloxymethyl and phthalidyl groups. The preferred carboxy protecting groups are allyl, benzyl and p-nitrobenzyl groups.
Suitable alkyl groups which R may represent include methyl, ethyl, propyl, butyl, pentyl and hexyl. Suitable alkenyl groups include allyl, propenyl, butenyl. Suitable phenylalkyl groups include benzyl and phenethyl.
As stated above, compounds of formula I may be converted into known antibacterial agents, as in detail explained and claimed in our published U.K. Patent Application GB 2111496-A and GB 2118181-A. These known antibacterial agents, named penems, are described for example in British Patent Specifications 2043639-B, 2097786 B and in the published European Application 0167100-A.
As described in the above cited prior art, the compounds of formula I are prepared by chemical selective hydrolysis of compounds of formula III: ##STR5## wherein R.sub.2 is as defined above, and X and Y are two different silyl derivatives such as t-butyldimethyl silyl and t-butyldiphenylsilyl groups respectively, in the presence of tetraalkylammonium fluoride.
The synthesis of compounds III and said selective removal of protecting group Y require expensive reagents and long reaction times which are not suitable for industrial large scale preparation of penems. The present invention provides a simple process for the preparation of compounds of the formula I by selective and inexpensive enzymatic hydrolysis of the compounds of formula II as defined above. The process of the invention, using enzymatic hydrolysis, allows the final product to be obtained under very mild conditions in very high yields and without undesired by-products.
The configuration of the compounds of formulae II and III is [5R,6S,(1R)], in order to obtain the preferred final [5R,6S,(1R)] stereochemistry of the penem nucleus. The starting materials of formula II are known compounds or may be prepared according to known procedures, for example as described in the published U.K. Patent Application GB 2144743-A.
Hydrolytic enzymes suitable for the present process include for example, lipases or proteases which selectively hydrolyze the carboxylic ester of the 2-hydroxymethyl residue (--CH.sub.2 OCOR) of the compound of formula (II) without affecting other functional groups which may be present. The hydrolytic process can be carried out either by using directly free or immobilized microbial cells which secrete a suitable enzyme or by isolating the specific enzymes which can be used in the free form, immobilized according to known techniques to resins, glass, cellulose or similar substances by ionic or covalent bonds, or grafted to fibres permeable to the substrate, or insolubilized by cross-linkage. Immobilization or insolubilization is advantageous as the same enzyme can be used for many production cycles. Moreover when an immobilized enzyme is used, the recovery of the reaction product is more easy. In fact, if the reaction product is absorbed on the resin at the end of the reaction, it is easily recovered in pure form by simply washing the resin with a suitable solvent.
The use of the enzymes isolated and purified to the desired degree is preferred rather than the raw cellular extract, since the extraction or purification process normally allows a reduction or elimination of the presence of contaminating enzymes which could lower the yields by formation of undesired by-products.
Also enzymatic preparations obtained by extraction of animal organs, such as porcine pancreas, are able to promote hydrolysis of the ester bond between the carbonyl group of an organic acid and the hydroxymethyl group in position 2 of the penem nucleus. Commercially available hydrolytic enzymes can be used in the hydrolytic process such as:
______________________________________ Enzyme Origin Seller ______________________________________ Pancreatin Porcine pancreas UNIBIOS - Trecate (Italy) Steapsin Porcine pancreas SIGMA Chem. Co. St. Louis (U.S.A.) Lipase Candida cylindracea SIGMA Chem. Co. St. Louis (U.S.A.) Lipase Wheat germ SIGMA Chem. Co. St. Louis (U.S.A.) Lipase SP 225 NOVO Industri (Denmark) Lipase Rhizopus Delamar SIGMA Chem. Co. St. Louis (U.S.A.) Lipase Chromobacterium TOYO JOZO (Japan) viscosum Lipoprotein Pseudomonas sp. TOYOBO (Japan) Lipase Protease Streptomyces SIGMA Chem. Co. caespitosus Protease Rhizopus sp. SIGMA Chem. Co. ______________________________________
The enzymes may be added to an aqueous suspension of from 1 to 100 g/l of the ester formula II, optionally containing small amounts of hydrocarbons, and suitably mildly buffered at different pH values, according to the enzyme used, which are in a range from 5 to 9, preferably from 6 to 8. The reaction may be carried out at a temperature of from 10.degree. C. to 50.degree. C., preferably from 20.degree. C. to 40.degree. C., for 0.5 to 48 hours, operating batchwise or in a column, according to the quantity of the enzyme present in the reaction mixture, and according to the ratio between the quantity of the enzyme in solution or in the immobilized form, and the quantity of substrate present in the reaction mixture. The pH of the reaction mixture is kept constant at the desired value by adding a solution of an alkali hydroxide thereto.
The yields of the reaction carried out under optimal conditions reach values higher than 90%. At the end of the reaction, the reaction product is recovered by conventional methods.
Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.